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Super engineering bacteria, expressed detoxification enzyme thereof, and construction method and application therefor

A technology for super-engineering and construction methods, applied in the fields of botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc.

Active Publication Date: 2010-10-06
辽宁中科生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] The purpose of the present invention is to solve the problem that my country has to use a large amount of pesticides as a large agricultural production country, and there is a serious pollution problem of pesticide residues in the environment. The loss caused by pesticide residue pollution to the national economy and the harm to people's health, thus providing a super engineering bacteria that can effectively degrade chemical pesticides

Method used

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  • Super engineering bacteria, expressed detoxification enzyme thereof, and construction method and application therefor
  • Super engineering bacteria, expressed detoxification enzyme thereof, and construction method and application therefor
  • Super engineering bacteria, expressed detoxification enzyme thereof, and construction method and application therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The cloning of the super engineered bacteria containing the genes of cytochrome oxidoreductase P450 CYP9G2 and cytochrome P450 reductase provided by the present invention is as follows:

[0053] 1.1 Plasmid and recipient bacteria

[0054] Cloning of Plutella xylostella CYP9G2 Gene

[0055] 1) Extraction of total RNA

[0056] 1-1) According to the operating procedures of the TRIzol kit, extract the total RNA of diamondback moth larvae: 2 RT-PCR reactions

[0057] 1-2) Perform a PCR reaction using the reverse-transcribed first-strand cDNA as a template, add the following reagents to the PCR reaction tube, and gently mix the reaction system;

[0058] 10×PCR Buffer 2.5μl

[0059] 50mM MgCl 2 0.75μl

[0060] 10mM dNTPs mixture 0.5μl

[0061] Forward primer (10μM) 0.5μl

[0062] Reverse primer (10μM) 0.5μl

[0063] cDNA 1μl

[0064] Taq DNA polymerase (5U / μl) 0.5μl

[0065] autoclaved distilled water 18.75μl

[0066] 1-3) The reaction program of the PCR reaction i...

Embodiment 2

[0126] Add 20ml of water and 1ml of 2% TritonX-100 to a 100ml Erlenmeyer flask, add an appropriate amount of ethanol solution of pesticides to make the final concentration of pesticides 10ppm, add 0.02M, pH 7.0 phosphate buffer to make up the volume, so that the total volume is 10mL. After fully mixing it, add 4 mL of the detoxifying enzyme crude enzyme solution prepared in Example 1 (approximately 30 mg of total protein), and shake on a shaker at 30°C-32°C. Samples were taken at 0min, 20min, 40min, 60min, 80min and 120min. Two repetitions were taken at each time point, and the CYP9G2 P450 single-expressing P450 9G2 enzyme prepared in Example 1 was used as the control group in the experiment. Take out 1ml of the treatment solution and put it in a 5ml test tube, add 1ml of redistilled n-hexane, shake it on the mixer for 3min to mix well, extract, then centrifuge at 6500rpm for 15min, take the supernatant, repeat the extraction once, and take out the supernatant After the liqui...

Embodiment 3

[0137] Add 20ml of water and 1ml of 2% TritonX-100 to a 100ml Erlenmeyer flask, add an appropriate amount of ethanol solution of pesticides, so that the final concentration of fenitrothion pesticide is 10ppm, add 0.02M, pH 7.0 phosphate buffer to make up the volume, so that the total The volume is 10 mL. After fully mixing it, add 4 mL of the detoxifying enzyme crude enzyme solution prepared in Example 1 (approximately 30 mg of total protein), and shake on a shaker at 30°C-32°C. Samples were taken at 0min, 20min, 40min, 60min, 80min and 120min. Two repetitions were taken at each time point, and the CYP9G2 P450 single-expressing P450 9G2 enzyme prepared in Example 1 was used as the control group in the experiment. Take out 1ml of the treatment solution and put it in a 5ml test tube, add 1ml of redistilled n-hexane, shake it on the mixer for 3min to mix well, extract, then centrifuge at 6500rpm for 15min, take the supernatant, repeat the extraction once, and take out the supern...

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Abstract

The invetnio discloses hyper-engineering bacteria to decompose residual pesticide with reserving number at CGMCC No.1529, which is characterized by the following: cloning the gene of P450 CYP9G2 and P450 reductase on the pETDuet-1; constructing pETDuet-CYP-POR; transmitting into escherichia coli; sieving to obtain the product. The invention also provides detoxication enzyme, which is fit for detoxicating organophosphorus intoxication animal.

Description

technical field [0001] The present invention relates to a super engineering bacterium and its construction and application, as well as the detoxification enzyme expressed by the super engineering bacterium and its application, in particular to a super engineering bacterium capable of degrading pesticide residues and the detoxification enzyme expressed by it, and Their construction methods and applications. Background technique [0002] Environment is the synthesis of human activity space and all surrounding elements, and is the premise and foundation of human development. The beauty of the environment is the main symbol of the quality of life and economic development. Resources are the basis for human survival and development, and the basic source of human means of production and living. We are entering the next 21st century, and China's modernization has entered a new stage. Can our resources and environment support the rapid development of the national economy? Will ra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/53A62D3/00C12N9/02C12R1/19C12N1/21
Inventor 乔传令申本昌兰文升丛健
Owner 辽宁中科生物工程股份有限公司
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